The outer-membrane Opa proteins of N. gonorrheae serve as bacterial adhesins. Opa proteins have a common structural motif, forming eight- stranded beta-barrels in the outer-membrane with four surface exposed loops. Three of the exposed loops consist of variable sequence domains (termed SV, HV-1 and HV-2) while a fourth loop, near the C-terminus of the protein, is highly conserved. Host-encoded receptors have been characterized for certain Opa proteins. The contribution of conserved versus variable sequence domains to the adherence mechanism is unknown. Our research objective is to characterize the behaviour of organisms expressing Opa proteins to determine whether the variable proteins represent true ?antigenic variants? (i.e. have the same biological phenotype and variable sequences to mask their recognition by the immune system) or are functional variants whose expression represents an adaptive response within the human host.A. Adherence to Heparan- Sulfate Proteoglycan (HSPG) ReceptorsCharacterization of the important Opa-protein domains in HSPG-receptor binding were studied using recombinant Opa proteins expressed from genetically modified opa genes in N. gonorrhoeae. These results showed that adherence was dependent on the presence of the OpaA HV-1 domain in that deletion of the region abrogated binding while transferring the region to a non-binding protein gave rise to novel HSPG-binding adhesins. Site-specific mutations in the region have identified a potential HSPG-binding motif. Experiments are in progress to determine whether peptide fragments or oligopeptides expressed as phage fusion proteins can block binding of live bacteria to human target cells.B. Adherence to CD66 ReceptorsThe basis for differential binding of Opa proteins by CD66 family members was studied using modified and recombinant versions of CD66. Modified glycoforms and fully deglycosylated CD66e molecules are recognized by Ng in an Opa-specific mannner, indicating that Opa proteins bind the N- domain peptide component of the receptor molecule. Homologue scanning mutagenesis was done using CD66e (binds all Opas) and CD66b (does not bind Opas) to determine which regions of the N-domain are important for adherence. Native, recombinant CD66b receptors were made by the incorporation of key amino acid changes that allowed for the binding of specific Opa proteins. Deletion and chimeric analysis of recombinant Opa proteins is presently underway to determine the role of the HV-1 and HV-2 variable domains in receptor binding. - Neisseria gonorrhoeae, Antigenic Variation, Opa, Bacterial Adhesins, Heparan Sulfate, Proteoglycans, CD66